The plastidial phosphorylase Pho1 co-localizes in situ with Photosystem I-associated PsaC as assessed by double labeling immunocytochemistry – transmission electron microscopy

The plastidial phosphorylase Pho1 is essential for normal starch biosynthesis in cereal grains. Biochemical studies, however, have shown that this starch biosynthetic enzyme interacts with PsaC, the terminal electron acceptor-donor protein of Photosystem I (PSI). This association with PsaC associated with PSI is supported by the localization of Pho1 to thylakoid membranes enriched in PSI complexes. To obtain direct in situ evidence for this protein-protein interaction within thylakoid membranes, we employed double-label immunocytochemistry at the electron microscopy level. Thin sections of leaf tissue from transgenic rice lines expressing GFP-tagged PsaC, as well as from wild-type plants, were incubated with mouse anti-GFP and rabbit anti-Pho1 antibodies, followed by secondary labeling with 6 nm and 12 nm gold particles, respectively, to visualize the antigen-antibody complexes. Despite the inherently low efficiency of immunogold labeling due to reduced antigenicity from chemical fixation and limited antibody penetration in resin-embedded tissue, we observed paired gold particles-at frequencies significantly higher than random-in which Pho1 was located in close proximity to GFP-PsaC. These findings support the direct interaction of Pho1 with PSI bound PsaC and, therefore, provide further justification to investigate the structural and functional basis of Pho1's influence on PSI activity.